Journal: The Journal of Biological Chemistry

Article Title: Structure-Function Analysis of VPS9-Ankyrin-repeat Protein (Varp) in the Trafficking of Tyrosinase-related Protein 1 in Melanocytes *

doi: 10.1074/jbc.M110.191205

Figure Lengend Snippet: All four ANK repeats are required for the Rab32/38 binding activity of the Varp ANKR1 domain. A, schematic representation of the truncated mutants of the ANKR1 domain of Varp used in this study. ANKR1-1 contains amino acid residues 462–494; ANKR1-2 contains amino acid residues 495–527; ANKR1-3 contains amino acid residues 528–560; and ANKR1-4 contains amino acid residues 561–596. ANKR1-Δ1 lacks amino acid residues 462–494 (Δ462–494); ANKR1-Δ2 lacks amino acid residues 495–527 (Δ495–527); ANKR1-Δ3 lacks amino acid residues 528–560 (Δ528–560); and ANKR1-Δ4 lacks amino acid residues 561–596 (Δ561–596). B, yeast two-hybrid assays revealed that all four ANK repeats in the ANKR1 are required for Rab32/38 binding. Yeast cells containing pAct2 plasmid expressing each Varp-ANKR1 mutant and pGBD plasmid expressing Rab32 mutant (left panels) or Rab38 mutant (right panels) were streaked on SC-LW (top panels) and SC-AHLW (bottom panels) and incubated at 30 °C. Note that none of the ANKR1 deletion mutants of Varp grew on SC-AHLW (i.e. selection medium) but that they grew normally on SC-LW.

Article Snippet: Sequence alignment of the switch II region of mouse or human Rabs or of each ANK repeat in the ANKR1 domain of Varp was performed by GENETYX-MAC (version 15.0.1; GENETYX Corp., Tokyo, Japan).

Techniques: Binding Assay, Activity Assay, Plasmid Preparation, Expressing, Mutagenesis, Incubation, Selection

Journal: The Journal of Biological Chemistry

Article Title: Structure-Function Analysis of VPS9-Ankyrin-repeat Protein (Varp) in the Trafficking of Tyrosinase-related Protein 1 in Melanocytes *

doi: 10.1074/jbc.M110.191205

Figure Lengend Snippet: Identification of the critical residues responsible for Rab32/38 binding in the ANKR1 domain of Varp by site-directed mutagenesis. A, sequence alignment of the ANKR1 domain of human (Hs, Homo sapiens), mouse (Mm, Mus musculus), chick (Gg, Gallus gallus), zebra fish (Dr, Danio rerio), sea urchin (Sp, Strongylocentrotus purpuratus), and silkworm (Bm, Bombyx mori) Varp. Residues conserved in more than five of the sequences are shown against a black background. The asterisks indicate the positions of the seven highly conserved amino acids, Gln-509, Cys-544, Lys-546, Tyr-550, Arg-557, Trp-575, and Tyr-577, in the ANKR1 domain that were the focus of the Ala-based site-directed mutagenesis. B, yeast two-hybrid assays revealed that the Gln-509 and Tyr-550 of Varp are critical for Rab32/38 binding. Yeast cells containing pGAD plasmid expressing Varp and pGBD plasmid expressing Rab32 (left panels) or Rab38 (right panels) were streaked on SC-LW (top panels) and SC-AHLW (selection medium; bottom panels) and incubated at 30 °C. Note that the Q509A, Y550A, W575A, and Y577A mutations dramatically reduced Rab32/38 binding activity, although the former two mutations reduced Rab32/38 binding activity more severely than the latter two mutations, based on the growth rate of the yeast cells and the results of the immunofluorescence analysis (see Fig. 6 and supplemental Fig. S4A). By contrast, the C544A, K546A, and R557A mutations had little or no effect on Rab32/38 binding. C, FLAG-tagged Rab32/38 and T7-tagged Varp-full or their mutants were co-expressed in COS-7 cells, and their associations were analyzed in the presence of 0.5 mm GTPγS by co-immunoprecipitation assays with anti-FLAG tag antibody-conjugated agarose beads as described previously (24). Co-immunoprecipitated T7-tagged Varp-full (or their mutants) (middle panels) and immunoprecipitated FLAG-tagged Rab32/38 (bottom panels) were detected with HRP-conjugated anti-FLAG tag antibody and HRP-conjugated anti-T7 tag antibody, respectively. Input means 1/70 volume of the reaction mixture used for immunoprecipitation (IP) (top panels). The positions of the molecular mass markers (in kilodaltons) are shown on the left. Note that neither Varp(Q509A) nor Varp(Y550A) bound Rab32/38 (lanes 3 and 4 in the middle panel), whereas Varp(R557A) normally bound Rab32/38 (lane 5 in the middle panel), consistent with the results of the yeast two-hybrid assays shown in B.

Article Snippet: Sequence alignment of the switch II region of mouse or human Rabs or of each ANK repeat in the ANKR1 domain of Varp was performed by GENETYX-MAC (version 15.0.1; GENETYX Corp., Tokyo, Japan).

Techniques: Binding Assay, Mutagenesis, Sequencing, Plasmid Preparation, Expressing, Selection, Incubation, Activity Assay, Immunofluorescence, Immunoprecipitation, FLAG-tag